2008 Glucose_6_Phosphate Dehydrogenase Deficiency Enhances Human Coronavirus 229E Infection PDF
Preview 2008 Glucose_6_Phosphate Dehydrogenase Deficiency Enhances Human Coronavirus 229E Infection
Glucose-6-Phosphate Dehydrogenase Deficiency Enhances Human Coronavirus 229E Infection Yi-Hsuan Wu,1 Ching-Ping Tseng,1,2,3 Mei-Ling Cheng,2,3 Hung-Yao Ho,2 Shin-Ru Shih,1,2,3 and Daniel Tsun-Yee Chiu1,2,3 1Graduate Institute of Basic Medical Sciences and 2Graduate Institute of Medical Biotechnology, Chang Gung University, and 3Department of Clinical Pathology, Chang Gung Memorial Hospital, Kwei-Shan, Tao-Yuan, Taiwan The host cellular environment is a key determinant of patho- gen infectivity. Viral gene expression and viral particle pro- duction of glucose-6-phosphate dehydrogenase (G6PD)–defi- cientandG6PD-knockdowncellsweremuchhigherthantheir counterparts when human coronavirus (HCoV) 229E was ap- plied at 0.1 multiplicity of infection. These phenomena were correlated with increased oxidant production. Accordingly, ectopic expression of G6PD in G6PD-deficient cells or addi- tion of antioxidant (such as �-lipoic acid) to G6PD- knockdown cells attenuated the increased susceptibility to HCoV 229E infection. All experimental data indicated that ox- idative stress in host cells is an important factor in HCoV 229E infectivity. Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzyme deficiency worldwide [1]. Accumulating evidence indicates that G6PD deficiency affects cells other than those of erythrocytes. For example, G6PD deficiency is now known to affect oocyte development and/or survival as well as growth and senescence of human foreskin fibroblasts (HFFs). Moreover, human G6PD-deficient neutrophils exhibit impaired production of nitric oxide, superoxide, and hydrogen peroxide, which may explain the impaired bactericidal effect of these cells [2]. Taken together, these findings indicate that G6PD defi- ciency causes abnormal cellular redox, thus affecting cells other than red cells. Oxidative stress is known to affect viral prolifera- tion and virulence [3]; however, the effect of G6PD deficiency on viral infectivity has not been thoroughly studied. In addition to being major sources of intracellular oxidants, pulmonary cells are exposed to �8000 L of oxygen-rich air daily as well as toxic particles [4]. Recent studies indicate that diesel exhaust exacerbates influenza virus infections in respiratory ep- ithelial cells [5]. Human coronavirus (HCoV) 229E, a common pathogen for respiratory tract infection, is a large, enveloped RNA virus and has a high affinity with airway cells. Recent iden- tification of a novel coronavirus as a causative agent of severe acute respiratory syndrome (SARS) has received substantial clinical attention. Because pulmonary cells are under high oxi- dative stress and because HCoV 229E is an interesting viral pathogen affecting pulmonary cells, it would be of interest to investigate how oxidative stress affects HCoV 229E infection of airway cells. Methods. Dulbecco’s modified Eagle medium (DMEM), trypsin, penicillin, streptomycin, Lipofectamine 2000 transfec- tion reagents, and dichlorofluorescin (DCF) diacetate were pur- chased from Invitrogen. The G6PD antibody was acquired from Genesis Biotech (Taiwan). Anti-actin and anti-CD13 antibodies were purchased from Santa Cruz Biotechnologies, and antibiotic G418 sulfate and �-lipoic acid were purchased from Promega. G6PD-deficient (HFF1), normal (HFF3), and G6PD-over- expressing fibroblasts (LGIN and LKGIN) were prepared as de- scribed elsewhere [6]. Lung carcinoma cells (A549) and human lung fibroblasts (MRC-5) were obtained from the American Type Culture Collection. All cells were cultured in DMEM sup- plemented with 10% fetal calf serum, 100 U/mL penicillin, and 100U/mL streptomycin at 37°C in a humidified atmosphere of 5% CO2 with or without 300 �g/mL G418, depending on whether or not they were transfected. For G6PD-RNAi plasmids, the complementary oligonucleo- tides G6PD-143S (5'-ACACACATATTCATCATCGAAGCTT- GGATGATGAATATGTGTGT-3') and G6PD–143AS (5'-GGA- TACACACATATTCATCATCCAAGCTTCGATGATGAATA- TGTGTGT-3') were annealed and ligated into pTOPO-U6 to generate pTOPO G6PD-143. The plasmid was tested and then removed to insert into the pCI-neo mammalian expression vec- tor, as described elsewhere [7]. Received 2 July 2007; accepted 1 October 2007; electronically published 12 February 2008. Potential conflicts of interest: none reported. Presented in part: Experimental Biology 2007, Washington, DC, 28 April–2 May 2007 (abstract LB323). Financial support: Chang Gung University (grant CMRPD140041); National Science Council of Taiwan (grant NSC94–2320-B182–041); Ministry of Education of Taiwan (grant EM- RPD150241). Reprints or correspondence: Dr. Shin-Ru Shih, Graduate Institute of Basic Medical Sciences, Chang Gung University, 259 Wen-Hwa First Rd., Kwei-Shan, Tao-Yuan, Taiwan (
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